Stability of liver-specific functions in hepatocytes is critical to the success of hepatocyte-based therapies for liver disease. The limitations in our current understanding of relevant hepatocellular cues are, in part, due to the structural complexity of the liver microarchitecture- repeating units with zones of hepatocyte specialization, gradients of soluble factors, interactions with extracellular matrix and non-parenchymal cells in 3-dimensions, and so on. The importance of each of these structural features on liver function has been widely emphasized; however, because it has historically been difficult to modulate these cues independently from each other, it remains unclear how they cooperate to exert their influence on hepatocellular function. The investigator has previously used micropatterning techniques to show that the balance between homotypic and heterotypic cell-cell interactions is a critical feature of tissue microarchitecture that dictates liver-specific function. It has now become evident that other factors in the microenvironment conspire to modulate the phenotype of hepatocytes including: oxygen concentration, growth factors, extracellular matrix, and 2- versus 3-dimensionality. The working hypothesis underlying the present proposal is that tissue microarchitecture influences zonal hepatocyte function through the interplay of soluble cues (O2, growth factors) and adhesive cues (cells, ECM) in the hepatocyte microenvironment. The general goal of this RENEWAL proposal is therefore to relate tissue microarchitecture to hepatocyte function by identifying and characterizing the mechanisms by which acellular cues in the local microenvironment cooperate to modulate hepatocyte function. Towards this end, the specific aims of this proposal are: (1) To investigate the cooperation of soluble factors (O2, growth factors, and paracrine signals) in regulating zonal hepatocyte function, (2) To investigate the cooperation of adhesive factors (ECM, cells) and soluble factors in regulating zonal function, and (3) To examine the role of soluble and adhesive factors in regulating zonal hepatocyte responses in 3-D microenvironments. In order to accomplish these goals, the investigator has established a research program where modern biological tools are integrated with several enabling technological platforms. A clear picture of the interplay between soluble and adhesive cues in 2- and 3-dimensions will be fundamental to the future design and implementation of hepatocyte-based therapies to treat liver disease.